Journal: RMD Open

Article Title: IL-6-PAD4 axis in the earliest phase of arthritis in knock-in gp130F759 mice, a model for rheumatoid arthritis

doi: 10.1136/rmdopen-2018-000853

Figure Lengend Snippet: PAD4 + cells in the synovium of gp130F759 are neutrophils. (A) Amounts of PAD4 protein and STAT3 phosphorylation in the wrist joints from wild type (WT) and gp130F759. Lysates of the joints from three mice of each genotype were pooled and subjected to western blotting. (B) Frozen sections of the synovium from WT and gp130F759 were stained with anti-PAD4 and pSTAT3 antibodies and Cy3-labelled F(ab’) 2 donkey anti-rabbit IgG (H+L). Representative pictures from four independent experiments are shown. Nuclei were stained with Hoechst 33342. Colocalisation was examined with anti-PAD4 and biotinylated pSTAT3 antibodies, which were visualised with Cy3-labelled anti-Rabbit IgG and Alexa488-streptavidin, respectively. A PAD4 producing cell whose STAT3 is phosphorylated (inset). The bars indicate 20 µm. (C) Gene expression levels of Padi4 and Il6 in haematopoietic (CD45 + ) or non-haematopoietic (CD45 − ) synovial cells which were separated with rat anti-mouse CD45 antibody and sheep anti-rat IgG magnetic beads. Summarised data from three independent experiments are shown. (D) IL-6 concentration in supernatant of primary culture of CD45 − synovial cells from WT and gp130F759 at 5 months old (n=7). *p<0.05. (E) Morphology of synovial cells producing PAD4. The photos of representative cytospin specimens from WT and gp130F759 are shown. Synovial cells cytospun onto the slide glass were incubated with anti-PAD4 antibody (red) and Hoechst 33342 for nuclear staining (blue). In the graph, black bar shows average and the open circle shows individual value for each mouse. (F) IL-6 produced by CD45 − synovial cells induced Padi4 expression in neutrophils. WT bone marrow neutrophils were stimulated for 6 hours with culture supernatant fluid (CSF) from gp130F759 pretreated with anti-IL-6 antibody or control IgG1. Then RNA from the neutrophils were prepared and transcription of Padi4 was estimated by real-time PCR using specific primers and SYBR green. Relative expression levels compared with Actb are shown. (G) IL-6 in the CSF from gp130F759 induced activation of STAT3 in neutrophils. WT bone marrow neutrophils were stimulated for 30 min with CSF from gp130F759 pretreated with anti-IL-6 antibody or control IgG1. The neutrophils were cyto-spun, air-dried and stained with anti-pSTAT3 antibody and Cy3-labelled donkey anti-rabbit IgG antibody. Pictures were taken with LSM700 confocal microscope. The bars indicate 10 µm.

Article Snippet: Anti-pSTAT3 antibody was stripped with Western BLoT Stripping Buffer (TAKARA Bio, Shiga, Japan), and then the membrane was reprobed with rabbit anti-STAT3 antibody (Cell Signaling Technology).

Techniques: Western Blot, Staining, Expressing, Magnetic Beads, Concentration Assay, Incubation, Produced, Real-time Polymerase Chain Reaction, SYBR Green Assay, Activation Assay, Microscopy

Journal: Oncotarget

Article Title: Tumor-associated macrophages promote neuroblastoma via STAT3 phosphorylation and up-regulation of c-MYC

doi: 10.18632/oncotarget.21066

Figure Lengend Snippet: (A) Mean fold change in percent of BrdU-positive NBT2 cells treated with solvent or JAK1/2 inhibitors AZD1480 (AZD) or ruxolitinib at indicated concentrations for NBT2 cells either alone or in co-culture with macrophages. Data were compiled from at least 3 independent experiments in triplicates; (B) Effects of AZD1480 and ruxolinitib on pSTAT3, STAT3, and MYC protein levels in lysates of NBT2 cells without or with a 6 and 24 hour co-culture with syngeneic macrophages in presence of AZD1480 (5 μM) or ruxolitinib (1 μM) as assessed by immunoblotting; (C) Immunoblot analysis of pSTAT3, STAT3, and MYC levels in protein lysates of three low MYC-expressing human NBL cell lines (LAN-6, CHLA-172, and CHLA-79) after 24 hour co-cultures with macrophages without or with ruxolitinib (1 μM); (D) Immunoblot analysis of STAT3 and pSTAT3 levels in protein lysates of tumors from NB-Tag mice or of subcutaneous tumors (Sub-Q) growing in NSG mice treated with ruxolitinib (60 mg/kg) by oral gavage twice daily for one week. (E) Tumor growth in NSG mice injected subcutaneously with 1X10 6 NBT2 tumor cells alone in one shoulder or co-injected in the opposite shoulder with equal number of macrophages that were conditioned for 24-36 hours with NBT2 cells in the transwell system. Animals were administered ruxolitinib (60 mg/kg) or drug vehicle by oral gavage twice daily for 3 weeks [ANOVA p < 0.005 between the untreated group (n = 12) and the treated group (n = 6); no significant difference was observed between treatment groups].

Article Snippet: Macrophages, TH levels and pSTAT3 were detected using rat anti-mouse F4/80 (Invitrogen, Carlsbad, CA: MF48000), unlabelled rabbit anti-mouse tyrosine hydroxylase polyclonal Ab (Abcam, Cambridge, UK: ab112) and rabbit unlabeled rabbit anti-mouse pSTAT3 (Tyr705) mAb (clone D3A7, Cell Signaling Technology, Danvers, MA), respectively.

Techniques: Solvent, Co-Culture Assay, Western Blot, Expressing, Injection

Journal: Oncotarget

Article Title: Tumor-associated macrophages promote neuroblastoma via STAT3 phosphorylation and up-regulation of c-MYC

doi: 10.18632/oncotarget.21066

Figure Lengend Snippet: (A) Immunoblot analysis showing STAT3 expression and phosphorylated STAT3 (pSTAT3) levels over time in protein lysates of NBT2 cells cultured in transwells with and without syngeneic murine macrophages. GAPDH is used as control for protein loading; (B) STAT3 expression and pSTAT3 levels assessed by immunoblotting protein lysates from adrenal glands of WT, NB-Tag, and NB-Tag- IL6 KO mice (14-22 weeks of age); (C) Representative images of pSTAT3 IHC in tumors of NB-Tag and NB-Tag- IL6 KO mice (inset: WT adrenal gland); (D) Immunoblots of STAT3 and pSTAT3 levels in NBT2 (murine) and CHLA-255 (human) NBL cells at basal level, and in the presence of IL-6 (10 ng/ml) or sIL-6R (25 ng/ml) either alone or with macrophages previously conditioned with tumor cell media, and incubated with IgG (control) or species-specific neutralizing anti-IL-6 mAb (1 and 5 μg/ml).

Article Snippet: Macrophages, TH levels and pSTAT3 were detected using rat anti-mouse F4/80 (Invitrogen, Carlsbad, CA: MF48000), unlabelled rabbit anti-mouse tyrosine hydroxylase polyclonal Ab (Abcam, Cambridge, UK: ab112) and rabbit unlabeled rabbit anti-mouse pSTAT3 (Tyr705) mAb (clone D3A7, Cell Signaling Technology, Danvers, MA), respectively.

Techniques: Western Blot, Expressing, Cell Culture, Control, Incubation

Journal: Cell

Article Title: Human MCTS1-dependent translation of JAK2 is essential for IFN-γ immunity to mycobacteria

doi: 10.1016/j.cell.2023.09.024

Figure Lengend Snippet: Key resources table

Article Snippet: Human pSTAT3-PE (pY705) , BD , Cat# 612569, RRID:AB_399860.

Techniques: Virus, Recombinant, Staining, Selection, Polymerase Chain Reaction, Plasmid Preparation, Reporter Assay, Sequencing, Mutagenesis, Variant Assay, Clone Assay, Knock-Out, Software, Next-Generation Sequencing

Journal: Nature Immunology

Article Title: The IL-22–oncostatin M axis promotes intestinal inflammation and tumorigenesis

doi: 10.1038/s41590-025-02149-z

Figure Lengend Snippet: a , H. hepaticus + anti-IL-10R colitis was induced in wild-type mice for 14 days. Protein analysis of IECs from steady-state ( n = 4) and H. hepaticus + anti-IL-10R ( n = 5) colitic mice was conducted. The western blot depicts phosphorylated STAT1 (pSTAT1), STAT1, pSTAT3, STAT3 and β-actin for representative samples from two experiments. b , c , Immunofluorescence staining for pSTAT1 and pSTAT3 in steady-state ( n = 7) and inflamed ( n = 10) mouse samples ( b ) with subsequent quantification ( c ) in the epithelium; scale bar, 20 μm. Hpf, high-power field. Data were pooled from two experiments. d , e , H. hepaticus + anti-IL-10R colitis was induced in wild-type mice for 7 days, and mice were treated with anti-IL-22 (clone 8e11, n = 4) or isotype control (mouse IgG1, GP120:9709, n = 5) on days 0 and 3 of colitis induction. Protein analysis of IECs from steady-state and H. hepaticus + anti-IL-10R colitic mice and anti-IL-22 or isotype-treated inflamed mice was performed. d , Western blot depicting STAT3 phosphorylation in epithelial cells from anti-IL-22- or isotype-treated colitic mice. Data are representative of two independent experiments. e , Quantification of STAT3 phosphorylation using ImageJ. The relative band intensity of blots from inflamed mice was normalized to that of steady-state untreated mice. f , g , Colitis was induced in Vil creERT2 Stat3 fl/fl (IEC Δ Stat3 ) or Vil creERT2 Stat3 fl/wt (IEC WT ; f ) and Stat1 –/– or Stat1 +/+ ( g ) mice using the H. hepaticus + anti-IL-10R model for 7 days. qPCR analysis was performed to assess the expression of Osmr and Reg3g in epithelial cells from inflamed mice normalized to epithelial cells isolated from untreated respective control mice. Data are representative of one experiment; n = 5–7 mice per genotype. h , Mouse colon organoids generated from Vil cre+ Stat3 fl/fl (IEC Δ Stat3 ) or Vil cre- Stat3 fl/fl (IEC WT ) mice and stimulated with 10 ng ml –1 IL-22. Relative expression of Osmr was analyzed by qPCR. Data are representative of three independent experiments from three independent biological replicates. P values (two-tailed) were calculated using a Mann–Whitney test for c and e – h .

Article Snippet: HCA-7 cells were also stimulated with 100 ng ml –1 recombinant human IL-22 or recombinant human OSM for 30 min, 24 h or 48 h, followed by analysis via MSD pSTAT3 assay (Meso Scale Diagnostics).

Techniques: Western Blot, Immunofluorescence, Staining, Control, Phospho-proteomics, Expressing, Isolation, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Immunology

Article Title: The IL-22–oncostatin M axis promotes intestinal inflammation and tumorigenesis

doi: 10.1038/s41590-025-02149-z

Figure Lengend Snippet: ( a ) Immunofluorescence staining for pSTAT1 and pSTAT3 in steady-state (n = 7) and inflamed (n = 10) mouse samples with subsequent quantification in colon lamina propria. Data pooled from two experiments. P -values are from Mann-Whitney U tests. ( b ) Western blot analysis of pSTAT3, total STAT3, and β-actin in mouse colon organoids treated with the indicated cytokines. Data are representative of two independent experiments. ( c ) Western blot analysis of pSTAT1, total STAT1, and β-actin in mouse colon organoids treated with indicated cytokines. Data are representative of two independent experiments. ( d ) Western blot analysis of pSTAT1, total STAT1, and β-actin in the human Caco2 cell line (left panel) was performed to confirm functionality of pSTAT1 antibody. Epithelial cells from C57BL/6 J mice with induced inflammation at days 7 and 14 (n = 5) were also analyzed (right panel). pSTAT1, total STAT1, and β-actin in steady state epithelial cells from C57BL/6 J mice are shown in Fig. . ( e ) Colon epithelial organoids isolated from C57BL/6 mice were treated with JAK/STAT pathway inhibitors (50 μM fludarabine, 25 μM STA-21, 2.5 μM ruxolitinib, or 2.5 μM tofacitinib) for 6 h, and 10 ng/mL of IL-22 was added for another 6 h. 0.1% DMSO was used as inhibitor control. Relative expression of Osmr was analyzed by qPCR. Data are shown for n = 2 biological replicates with n = 3 technical replicates. Graph displays mean ± SEM. P -values are derived from Kruskal-Wallis test.

Article Snippet: HCA-7 cells were also stimulated with 100 ng ml –1 recombinant human IL-22 or recombinant human OSM for 30 min, 24 h or 48 h, followed by analysis via MSD pSTAT3 assay (Meso Scale Diagnostics).

Techniques: Immunofluorescence, Staining, MANN-WHITNEY, Western Blot, Isolation, Control, Expressing, Derivative Assay

Journal: Nature Immunology

Article Title: The IL-22–oncostatin M axis promotes intestinal inflammation and tumorigenesis

doi: 10.1038/s41590-025-02149-z

Figure Lengend Snippet: ( a ) Human HCA-7 colon epithelial cells were stimulated with 100 ng/ml of rhIL-22 or rhOSM for 30 min, 24 or 48 h. Subsequently, pSTAT3 was quantified in cell lysates using an electrochemiluminescence assay (MSD). Data are representative of two independent experiments, each with three technical replicates. P -values derived from a 2-way comparison using Tukey’s multiple comparison test. ( b ) Analysis of STAT3 phosphorylation after colitis induction in IECs from IEC Δ Osmr and control mice (values normalized to those from steady-state mice). P -values are derived from Mann–Whitney U test. Data are representative of two independent experiments; n = 6 mice per group. ( c ) Volcano plot depicting differentially expressed genes in the epithelial cell line, HCA-7, after treatment with rhOSM for 24 h as described in ( a ). ( d ) GSEA plot depicting enrichment of OSM-activated genes in epithelial cells (defined in analysis shown in panel c ) comparing inflamed IEC Δ Osmr and control mice, as in Fig. .

Article Snippet: HCA-7 cells were also stimulated with 100 ng ml –1 recombinant human IL-22 or recombinant human OSM for 30 min, 24 h or 48 h, followed by analysis via MSD pSTAT3 assay (Meso Scale Diagnostics).

Techniques: Electrochemiluminescence, Derivative Assay, Comparison, Phospho-proteomics, Control, MANN-WHITNEY

Journal: Nature Immunology

Article Title: The IL-22–oncostatin M axis promotes intestinal inflammation and tumorigenesis

doi: 10.1038/s41590-025-02149-z

Figure Lengend Snippet: a , Wild-type mice underwent AOM/DSS-induced CAC. Colon tissues from tumor-bearing mice were analyzed by ISH for Osmr (blue) and Pdgfra (red; stromal marker); scale bars, 1,000 μm (left) and 200 μm (two insets on the right). b , Spatial transcriptomics analysis of a biopsy from an individual with CAC, including tumor tissue (red box) and adjacent normal mucosa (green box). OSM and OSMR expression was assessed in epithelial and nonepithelial cells using epithelial ( EPCAM , CDH1 , KRT20 , MUC5B and MKI67 ) and nonepithelial markers ( CD3E , CD4 , CD14 , CD68 , C1QC , CD19 , CD79A , PDGFRA , VIM , PECAM1 , CD34 and KIT ); scale bar, 1,000 μm (left) and 100 μm (insets on the right). c , Quantification of OSMR expression in epithelial and nonepithelial cells in CAC ( n = 10, red bars) and healthy control ( n = 3, black bars) tissue, along with nonepithelial OSM expression. d , Experimental schematic comparing CAC induction in wild-type versus Osm –/– mice. Representative colonoscopy images and tumor burden on day 80 are shown ( n = 25 per genotype). e , Wild-type mice received AOM and DSS, followed by treatment with anti-OSM ( n = 28) or isotype antibody (rIgG2a; n = 26). Tumor burden, multiplicity and volume were assessed on day 80. Data are shown normalized to the isotype-treated group. f , Immunostaining for pSTAT3 in colon tumors and adjacent normal tissues from mice treated as in e ; scale bar, 200 μm. g , Quantification of pSTAT3 + epithelial cells ( n = 10 per group). h , Relative pSTAT3 expression intensity normalized to that observed in control tissue ( n = 10 per group). Data are derived from two experiments. P values (two-tailed) were calculated from one-sample t -tests and Wilcoxon rank tests. i , j , Vil creERT2+ Osmr fl/fl (IEC Δ Osmr ; n = 15) and Vil creERT2+ Osmr wt/wt (IEC WT ; n = 18) mice were treated with AOM/DSS to induce CAC. Tamoxifen administration induced epithelial-specific Osmr deletion. i , Experimental design and representative colon images. j , Tumor burden, multiplicity and average volumes from two independent experiments. Data are shown normalized to the Vil creERT2+ Osmr wt/wt group. P values (two-tailed) were calculated using a Mann–Whitney test in c – e , g and j .

Article Snippet: HCA-7 cells were also stimulated with 100 ng ml –1 recombinant human IL-22 or recombinant human OSM for 30 min, 24 h or 48 h, followed by analysis via MSD pSTAT3 assay (Meso Scale Diagnostics).

Techniques: Marker, Expressing, Control, Immunostaining, Derivative Assay, Two Tailed Test, MANN-WHITNEY

Journal: Cancers

Article Title: The PAK1-Stat3 Signaling Pathway Activates IL-6 Gene Transcription and Human Breast Cancer Stem Cell Formation

doi: 10.3390/cancers11101527

Figure Lengend Snippet: PAK1 physically interacts with Stat3, PAK1 and Stat3 colocalize in the nucleus and PAK1 inactivation induces Stat3 dephosphorylation. ( A ) Total protein from breast cancer cells was immunoprecipitated with anti-PAK1 or anti-Stat3 antibodies, followed by immunoblotting using anti-Stat3 or anti-PAK1 antibodies. ( B ) Immunofluorescence analysis of PAK1 (green) and Stat3 (red) expression in breast cancer cells using the indicated antibodies. Images were captured by microscopy at 10× magnification. ( C ) Effect of ivermectin on the pStat3 level. ( D ) Immunofluorescence analysis of PAK1 (green) and Stat3 (red) expression levels in breast cancer cells after ivermectin treatment. Images were captured by microscopy at 10× magnification (scale bar = 100 μm).

Article Snippet: The pPAK1 and pStat3 plasmids were purchased from Addgene (Watertown, MA, USA).

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Western Blot, Immunofluorescence, Expressing, Microscopy

Journal: Cancers

Article Title: The PAK1-Stat3 Signaling Pathway Activates IL-6 Gene Transcription and Human Breast Cancer Stem Cell Formation

doi: 10.3390/cancers11101527

Figure Lengend Snippet: Effect of a JAK2 inhibitor on nuclear PAK1 and Stat3 levels and on the colocalization of PAK1 and Stat3. ( A ) Immunofluorescence analysis of PAK1 (green) and Stat3 (red) expression in breast cancer cells using anti-PAK1 and anti-Stat3 antibodies after TG101209 treatment. Images were captured by microscopy at 10× magnification. ( B ) Effect of TG101209 on cytosolic and nuclear pStat3 protein levels.

Article Snippet: The pPAK1 and pStat3 plasmids were purchased from Addgene (Watertown, MA, USA).

Techniques: Immunofluorescence, Expressing, Microscopy

Journal: Cancers

Article Title: The PAK1-Stat3 Signaling Pathway Activates IL-6 Gene Transcription and Human Breast Cancer Stem Cell Formation

doi: 10.3390/cancers11101527

Figure Lengend Snippet: PAK1 affects IL-6 gene expression through Stat3-mediated regulation. ( A ) Electrophoretic mobility shift assay (EMSA) using mammosphere nuclear proteins. Nuclear proteins were incubated with an IRDye 800-Stat probe at room temperature and separated by 6% native PAGE. Lane 1: stat probe; lane 2: nuclear proteins with stat probe; lane 3: 10-fold concentration of the self-competitor oligo; lane 4: nuclear proteins incubated with a 10-fold concentration of the mutated-Stat3 probe. The arrow indicates the DNA/Stat3 complex in mammosphere nuclear lysates. ( B ) EMSA analysis of PAK1 and Stat3 binding to the Stat3 DNA-binding sequence. Lane 1: Stat probe only; lane 2: nuclear proteins with Stat probe; lane 3: 10-fold concentration of the self-competitor oligo; lane 4: 10-fold concentration of the mutated Stat probe; lane 5: pStat3 antibody; lane 6: PAK1 antibody; lane 7: pStat3 and PAK1 antibody; lane 8: IgG. The arrow indicates the Stat3/PAK1/DNA complex in nuclear proteins. ( C ) ChIP (chromatin immunoprecipitation) analysis of Stat3 and PAK1 recruitment to the IL-6 gene promoter region in breast cancer cells. Double-ChIP analysis of the Stat3-PAK1/DNA complex binding to the human IL-6 gene promoter in breast cancer cells. The first ChIP was performed with an anti-pStat3 or anti-PAK1 antibody, followed by a second ChIP with an anti-PAK1 or anti-pStat3 antibody, respectively. Input represents 1% of the sonicated DNA. ( D ) RT-qPCR analysis of human IL-6 mRNA levels in breast cancer cells transiently overexpressing PAK1 and Stat3 genes. The relative mRNA expression levels are presented. The data are presented as the mean ± SD; n = 3; * p < 0.05 vs. control vector. ( E ) Nuclear levels of Stat3 and NF-κB (p65) protein in mammospheres were examined with antibodies against Stat3 and p65. Ivermectin treatment reduced nuclear pStat3 levels in the mammospheres. ( F ) EMSA of lysates of mammospheres treated with ivermectin. Nuclear proteins were treated with an IRDye 800-Stat sequence probe and separated by 6% native PAGE. Lane 1: stat probe; lane 2: nuclear proteins with stat probe; lane 3: ivermectin-treated proteins with stat probe; lane 4: 10-fold concentration of self-competitor oligo; lane 5: 10-fold concentration of mutated Stat3 oligo. Ivermectin treatment decreases the DNA/Stat3 interaction in mammosphere nuclei. ( G ) Transcriptional levels of the human IL-6 gene were assayed in ivermectin-treated cells using IL-6 -specific primers. β-actin expression was used to normalize the values. The data are presented as the mean ± SD; n = 3; * p < 0.05 vs. control. ( H ) Immunoblot analysis of culture medium from mammospheres using an IL-6 antibody and equal numbers of mammospheres as a control. Ivermectin treatment reduced extracellular IL-6 levels in mammosphere culture medium. ( I ) The cytokine profiles of conditioned media from mammosphere cultures were determined with cytokine-specific antibodies and cytokine beads. Ivermectin reduced extracellular IL-6 levels in the mammosphere cultures.

Article Snippet: The pPAK1 and pStat3 plasmids were purchased from Addgene (Watertown, MA, USA).

Techniques: Gene Expression, Electrophoretic Mobility Shift Assay, Incubation, Clear Native PAGE, Concentration Assay, Binding Assay, Sequencing, Chromatin Immunoprecipitation, Sonication, Quantitative RT-PCR, Expressing, Control, Plasmid Preparation, Western Blot